STABILITY OF ESCHERICHIA-COLI PHOSPHOENOLPYRUVATE CARBOXYKINASE AGAINST UREA-INDUCED UNFOLDING AND LIGAND EFFECTS

The urea-induced unfolding at pH 7.5 of Escherichia coil phosphoenolpy ruvate (P-pyruvate) carboxykinase was studied by monitoring the enzyme activity, intrinsic protein fluorescence, circular dichroism spectra, and 1-anilino-8-naphthalenesulfonate binding. These studies were perf ormed in the absence and presence of substrates and ligands. ATP or P- pyruvate plus MnCl2, or of the combined presence of ATP plus MnCl2 and oxalate, conferred great protection against urea-induced denaturation . The unfolding process showed the presence of at least one stable int ermediate which is notably shifted to higher urea concentrations in th e presence of substrates. This intermediate protein structure was inac tive, contained less tertiary structure than the native protein and re tained mos: of the original secondary structure. Hydrophobic surfaces were more exposed in the intermediate than in the native or unfolded s pecies. Refolding experiments indicated that the secondary structure w as completely recovered. Total recovery of tertiary structure and acti vity was obtained only from samples denatured at urea concentrations l ower than those where the intermediate accumulates.